Characterization of proteins often requires the ability to incorporate detectable groups—e.g., fluorochromes, chromophores, spin labels, radioisotopes, paramagnetic atoms, heavy atoms, haptens, crosslinking agents, and cleavage agents—at specific, defined sites. For proteins that do not contain pre-existing cysteine residues, site-specific labeling can be accomplished by use of site-directed mutagenesis to introduce a cysteine residue at the site of interest, followed by cysteine-specific chemical modification to incorporate the labeled probe. However, for proteins that contain pre-existing cysteine residues, site-specific labeling is difficult. Multiple strategies have been reported: (i) intein-mediated labeling (“expressed protein ligation”), (Muir, et al., Proc. Natl. Acad. Sci. USA, 95:6705–6710 (1998)); (ii) transglutaminase-mediated labeling (Sato et al., Biochem. 35:13072–13080 (1996)); (iii) oxidation-mediated labeling (Geoghegan, et al., Bioconj. Chem., 3:138–146 (1992)); (iv) transition-metal-chelate-mediated labeling (Kapanidis et al., J. Amer. Chem. Soc., 123:12123 (2001)); and (v) trivalent-arsenic-mediated labeling (Griffin et al., Science 281:269–272, 1998) (U.S. Pat. No. 6,008,378). Strategies (i)–(iii) do not permit in situ labeling (i.e., direct labeling of proteins in cuvettes, gels, blots, or biological samples—without the need for a subsequent purification step) or in vivo labeling (i.e., direct labeling of proteins in living cells). Strategy (iv) does not permit labeling and analysis at subnanomolar concentrations. Strategy (v) requires a structural scaffold presenting two trivalent-arsenic atoms in a precisely defined spatial relationship and therefore relates only to a limited number of detectable groups (such as those having a xanthene, xanthanone, or phenoxazine structural nucleus).
There is a need for improved methods and compositions for protein labeling. In particular, there is a need for methods and compositions that permit in situ labeling, that permit in vivo labeling, that permit labeling and analysis at sub-nanomolar concentrations, and that encompass a wide range of detectable groups with different properties.